Edited by Peter Devreotes, Johns Hopkins University School of Medicine, Baltimore, MD; received December 13, 2022; accepted February 3, 2023
March 10, 2023
120 (11) e2220825120
Significance
Macroendocytosis, the uptake of extracellular material, is an actin-driven process crucial for the clearance of pathogens by macrophages and nutrient uptake in lower eukaryotes. Hitherto, in Dictyostelium cells, the Arp2/3 complex and VASP were implicated in actin assembly at the protrusive rim of cups, whereas the Ras-regulated formin ForG was shown to generate half of the F-actin mass at its supporting base, raising questions about additional factors contributing to actin filament formation at this site. Our analysis unexpectedly revealed the presence of a Rac-regulated formin (ForB) pathway that acts synergistically with the ForG pathway to promote filament assembly in the base. However, despite functional overlap of these formins, only ForB promotes actin-driven phagosomal movement, known as rocketing, after particle internalization.
Abstract
Macroendocytosis comprising phagocytosis and macropinocytosis is an actin-driven process regulated by small GTPases that depend on the dynamic reorganization of the membrane that protrudes and internalizes extracellular material by cup-shaped structures. To effectively capture, enwrap, and internalize their targets, these cups are arranged into a peripheral ring or ruffle of protruding actin sheets emerging from an actin-rich, nonprotrusive zone at its base. Despite extensive knowledge of the mechanism driving actin assembly of the branched network at the protrusive cup edge, which is initiated by the actin-related protein (Arp) 2/3 complex downstream of Rac signaling, our understanding of actin assembly in the base is still incomplete. In the Dictyostelium model system, the Ras-regulated formin ForG was previously shown to specifically contribute to actin assembly at the cup base. Loss of ForG is associated with a strongly impaired macroendocytosis and a 50% reduction in F-actin content at the base of phagocytic cups, in turn indicating the presence of additional factors that specifically contribute to actin formation at the base. Here, we show that ForG synergizes with the Rac-regulated formin ForB to form the bulk of linear filaments at the cup base. Consistently, combined loss of both formins virtually abolishes cup formation and leads to severe defects of macroendocytosis, emphasizing the relevance of converging Ras- and Rac-regulated formin pathways in assembly of linear filaments in the cup base, which apparently provide mechanical support to the entire structure. Remarkably, we finally show that active ForB, unlike ForG, additionally drives phagosome rocketing to aid particle internalization.
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Data, Materials, and Software Availability
All study data are included in the article and/or SI Appendix. Constructs and mutants can be obtained from the corresponding author ([email protected]) upon reasonable request.
Acknowledgments
We thank Annette Breskott for technical assistance. This work was supported by a grant of the Deutsche Forschungsgemeinschaft to J.F. (FA330/13-1).
Author contributions
S.K. and J.F. designed research; S.K. and J.F. performed research; A.J., C.L., and M.W. contributed new reagents/analytic tools; S.K. analyzed data; and S.K. and J.F. wrote the paper.
Competing interests
The authors declare no competing interest.
Supporting Information
Movie S1.
ForB co-localizes with F-actin at sites of macropinocytosis. Confocal time-lapse imaging of a WT cell expressing YFP-ForBΔDAD (green) and mRFP-LifeAct (magenta). Still images from this movie are shown in Fig. 1C. Time is in min:s. Scale bar, 5 μm.
Movie S2.
ForB localizes prominently to phagocytic cups. Confocal time-lapse imaging of a WT cell expressing YFP-ForBΔDAD (green) during internalization of a TRITC-labelled yeast particle (magenta). Still images from this movie are shown in Fig. 1F. Time is in min:s. Scale bar, 5 μm.
Movie S3.
ForB interacts with active RacB interact in vivo and localizes to sites of phagocytosis. Confocal timelapse imaging of a WT cell expressing VC-ForBΔDAD and VN-RacB Q61L (green), during internalization of a TRITC-labelled yeast particle (magenta). Still images from this movie are shown in Fig. 3C. Time is in min:s. Scale bar, 5 μm.
Movie S4.
ForB and active RacB interact in vivo and co-localize to macropinocytic cups. Confocal, time-lapse imaging of a WT cell expressing VC-ForBΔDAD and VN-RacB Q61L (green). Still images from this movie are shown in Fig. 3D. Time is in min:s. Scale bar, 5 μm.
Movie S5.
Overexpression of active ForB promotes the rocketing of internalized phagosomes. Confocal, time-lapse imaging of a WT cell expressing YFP-ForBΔDAD (green) during and after internalization of a TRITC-labelled yeast particle (magenta). Still images from this movie are shown in Fig. 5A. Time is in min:s. Scale bar, 5 μm.
Movie S6.
Confocal, time-lapse imaging of a WT cell expressing YFP-ForB-N (green) and mRFP-LifeAct (magenta) during internalization of an Atto633-labelled yeast particle (blue). Still images from this movie are shown in Fig. 5B. Time is in min:s. Scale bar, 5 μm.
References
1
A. Aderem, D. M. Underhill, Mechanisms of phagocytosis in macrophages. Year. Rev. Immunol. 17593–623 (1999).
2
J. A. Swanson, Shaping cups into phagosomes and macropinosomes. Nat. Rev. Mol. Cell Biol. 9639–649 (2008).
3
M. Rabinovitch, Professional and non-professional phagocytes: An introduction. Trends Cell Biol. 585–87 (1995).
4
J. J. Lim, S. Grinstein, Z. Roth, Diversity and versatility of phagocytosis: Roles in innate immunity, tissue remodeling, and homeostasis. Front. Cell Infect. Microbiol. 7191 (2017).
5
S. Arandjelovic, K. S. Ravichandran, Phagocytosis of apoptotic cells in homeostasis. Nat. Immunol. 16907–917 (2015).
6
J. D. Dunn et al., Eat prey, live: Dictyostelium discoideum as a model for cell-autonomous defenses. Front. Immunol. 81906 (2017).
7
W. F. Loomis, Dictyostelium discoideum, A Developmental System (Academic Press Inc., New York, 1975).
8
J. S. King, R. R. Kay, The origins and evolution of macropinocytosis. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 37420180158 (2019).
9
D. B. Mills, The origin of phagocytosis in Earth history. Interface Focus. 1020200019 (2020).
10
N. Yutin, M. Y. Wolf, Y. I. Wolf, E. v Koonin,, The origins of phagocytosis and eukaryogenesis. Biol. Direct 49 (2009).
11
J. Martín-González, J.-F. Montero-Bullón, J. Lacal, Dictyostelium discoideum as a non-mammalian biomedical model. Microb. Biotechnol. 14111–125 (2021).
12
M. J. Carnell, R. H. Insall, Acti—On disease–Studying the pathobiology of cell motility using Dictyostelium discoideum. Semin Cell Dev. Biol. 2282–88 (2011).
13
G. Bloomfield et al., Neurofibromin controls macropinocytosis and phagocytosis in Dictyostelium. Elife 4e04940 (2015).
14
R. Sussman, M. Sussman, Cultivation of Dictyostelium discoideum in axenic medium. Biochem. Biophys. Res. Commun. 2953–55 (1967).
15
J. H. Vines, J. S. King, The endocytic pathways of Dictyostelium discoideum. Int. J. Dev. Biol. 63461–471 (2019).
16
V. Jaumouillé, C. M. Waterman, Physical constraints and forces involved in phagocytosis. Front. Immunol. 111097 (2020).
17
D. M. Veltman et al., A plasma membrane template for macropinocytic cups. Elife 5e20085 (2016).
18
S. A. Freeman, S. Grinstein, Phagocytosis: Receptors, signal integration, and the cytoskeleton. Immunol. Rev. 262193–215 (2014).
19
E. Uribe-Querol, C. Rosales, Phagocytosis: Our current understanding of a universal biological process. Front. Immunol. 111066 (2020).
20
S. Cornillon et al., An adhesion molecule in free-living Dictyostelium amoebae with integrin β features. EMBO Rep. 7617–621 (2006).
21
M. Pan, X. Xu, Y. Chen, T. Jin, Identification of a chemoattractant G-protein-coupled receptor for folic acid that controls both chemotaxis and phagocytosis. Dev. Cell 36428–439 (2016).
22
M. B. Hallett, An introduction to phagocytosis. Adv. Exp. Med. Biol. 12461–7 (2020).
23
S. Buracco, S. Claydon, R. Insall, Control of actin dynamics during cell motility. F1000 Res 8F1000 Faculty Rev-1977 (2019).
24
D. J. Seastone et al., The WASp-like protein scar regulates macropinocytosis, phagocytosis and endosomal membrane flow in Dictyostelium. J. Cell Sci. 1142673–2683 (2001).
25
M. A. Wear, A. Yamashita, K. Kim, Y. Maéda, J. A. Cooper, How capping protein binds the barbed end of the actin filament. Curr. Biol. 131531–1537 (2003).
26
J. Faix, K. Rottner, Ena/VASP proteins in cell edge protrusion, migration and adhesion. J. Cell Sci. 135jcs259226 (2022).
27
D. Breitsprecher et al., Clustering of VASP actively drives processive, WH2 domain-mediated actin filament elongation. EMBO J. 272943–2954 (2008).
28
S. Körber, J. Faix, VASP boosts protrusive activity of macroendocytic cups and drives phagosome rocketing after internalization. Eur. J. Cell Biol. 101151200 (2022).
29
N. Courtemanche, Mechanisms of formin-mediated actin assembly and dynamics. Biophys. Rev. 101553–1569 (2018).
30
J. Block et al., FMNL2 drives actin-based protrusion and migration downstream of Cdc42. Curr. Biol. 221005–1012 (2012).
31
C. Litschko et al., Functional integrity of the contractile actin cortex is safeguarded by multiple Diaphanous-related formins. Proc. Natl. Acad. Sci. U.S.A. 1163594–3603 (2019).
32
T. D. Pollard, Regulation of actin filament assembly by Arp2/3 complex and formins. Annu. Rev. Biophys. Biomol. Struct. 36451–477 (2007).
33
A. Seth, C. Otomo, M. K. Rosen, Autoinhibition regulates cellular localization and actin assembly activity of the diaphanous-related formins FRLα and mDia1. J. Cell Biol. 174701–713 (2006).
34
Structural basis of Rho GTPase-mediated activation of the formin mDia1. Mol. Cell 18273–281 (2005).
35
P. Massol, P. Montcourrier, J. C. Guillemot, P. Chavrier, Fc receptor-mediated phagocytosis requires CDC42 and Rac1. EMBO J. 176219–6229 (1998).
36
A. D. Hoppe, J. A. Swanson, Cdc42, Rac1, and Rac2 display distinct patterns of activation during phagocytosis. Mol. Biol. Cell 153509–3519 (2004).
37
D. J. Hackam, O. D. Rotstein, A. Schreiber, W. j. Zhang, S. Grinstein, Rho is required for the initiation of calcium signaling and phagocytosis by Fcγ receptors in macrophages. J. Exp. Med. 186955–966 (1997).
38
A. L. Bishop, A. Hall, Rho GTPases and their effector proteins. Biochem. J. 348241–255 (2000).
39
D. M. Veltman, M. G. Lemieux, D. A. Knecht, R. H. Insall, PIP3-dependent macropinocytosis is incompatible with chemotaxis. J. Cell Biol. 204497–505 (2014).
40
A. Junemann et al., A Diaphanous-related formin links Ras signaling directly to actin assembly in macropinocytosis and phagocytosis. Proc. Natl. Acad. Sci. U.S.A. 113E7464–E7473 (2016).
41
F. Rivero et al., A comparative sequence analysis reveals a common GBD/FH3-FH1-FH2-DAD architecture in formins from Dictyosteliumetazoan and metazoa. BMC Genome. 628 (2005).
42
C. Kitayama, T. Q. P. Uyeda, ForC, a novel type of formin family protein lacking an FH1 domain, is involved in multicellular development in Dictyostelium discoideum. J. Cell Sci. 116711–723 (2003).
43
M. Ecke et al., Formins specify membrane patterns generated by propagating actin waves. Mol. Biol. Cell 31373–385 (2020).
44
H. Beug, F. E. Katz, G. Gerisch, Dynamics of antigenic membrane sites relating to cell aggregation in Dictyostelium discoideum. J. Cell Biol. 56647–658 (1973).
45
A. de Lozanne, J. A. Spudich, Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination. Science 2361086–1091 (1987).
46
C. Litschko, J. Damiano-Guercio, S. Brühmann, J. Faix, “Analysis of random migration of Dictyostelium amoeba in confined and unconfined environments BT” in Cell Migration: Methods and Protocols, A. Gautreau, Ed. (Springer, New York, 2018), pp. 341–350.
47
G. Vlahou, F. Rivero, Rho GTPase signaling in Dictyostelium discoideum: Insights from the genome. Eur. J. Cell Biol. 85947–959 (2006).
48
K. Ohashi, K. Mizuno, A novel pair of split venus fragments to detect protein-protein interactions by in vitro and in vivo bimolecular fluorescence complementation assays. Methods Mol. Biol. 1174247–262 (2014).
49
J. A. Theriot, T. J. Mitchison, L. G. Tilney, D. A. Portnoy, The rate of actin-based motility of intracellular Listeria monocytogenes equals the rate of actin polymerization. Nature 357257–260 (1992).
50
T. P. Loisel, R. Boujemaa, D. Pantaloni, M. F. Carlier, Reconstitution of actin-based motility of Listeria and Shigella using pure proteins. Nature 401613–616 (1999).
51
W. Witke, M. Schleicher, A. A. Noegel, Redundancy in the microfilament system: Abnormal development of Dictyostelium cells lacking two F-actin cross-linking proteins. Cell 6853–62 (1992).
52
J. Faix et al., Cortexillins, major determinants of cell shape and size, are actin-bundling proteins with a parallel coiled-coil tail. Cell 86631–642 (1996).
53
T. Bretschneider et al., The three-dimensional dynamics of actin waves, a model of cytoskeletal self-organization. Biophys. J. 962888–2900 (2009).
54
W. O. Fenn, The adhesiveness of leucocytes to solid surfaces. J. Gen. Physiol. 5143–167 (1922).
55
G. Gerisch, Self-organizing actin waves that simulate phagocytic cup structures. PMC Biophys. 37 (2010).
56
F. Rivero, H. Dislich, G. Glöckner, A. A. Noegel, The Dictyostelium discoideum family of Rho-related proteins. Nucleic Acids Res. 291068–1079 (2001).
57
M. Dumontier, P. Höcht, U. Mintert, J. Faix, Rac1 GTPases control filopodia formation, cell motility, endocytosis, cytokinesis and development in Dictyostelium. J. Cell Sci. 1132253–2265 (2000).
58
F. Rivero et al., RacF1, a novel member of the Rho protein family in Dictyostelium discoideum, associates transiently with cell contact areas, macropinosomes, and phagosomes. Mol. Biol. Cell 101205–1219 (1999).
59
E. Lee, D. J. Seastone, E. Harris, J. A. Cardelli, D. A. Knecht, RacB regulates cytoskeletal function in Dictyostelium spp. Eukaryot Cell 2474–485 (2003).
60
K. C. Park et al., Rac regulation of chemotaxis and morphogenesis in Dictyostelium. EMBO J. 234177–4189 (2004).
61
S. Mondal et al., Regulation of the actin cytoskeleton by an interaction of IQGAP related protein GAPA with filamin and cortexillin I. PLoS One 5e15440 (2010).
62
S. Lee et al., Dictyostelium PAKc is required for proper chemotaxis. Mol. Biol. Cell 155456–5469 (2004).
63
M. de la Roche, A. Mahasneh, S.-F. Lee, F. Rivero, G. P. Côté, Cellular distribution and functions of wild-type and constitutively activated Dictyostelium PakB. Mol. Biol. Cell 16238–247 (2005).
64
C. M. Buckley et al., Coordinated Ras and Rac activity shapes macropinocytic cups and enables phagocytosis of geometrically diverse bacteria. Curr. Biol. 302912–2926.e5 (2020).
65
M. Marinović et al., IQGAP-related protein IqgC suppresses Ras signaling during large-scale endocytosis. Proc. Natl. Acad. Sci. U.S.A. 1161289–1298 (2019).
66
P. Chugh, E. K. Paluch, The actin cortex at a glance. J. Cell Sci. 131jcs186254 (2018).
67
M. Bovellan et al., Cellular control of cortical actin nucleation. Curr. Biol. 241628–1635 (2014).
68
M. Fritzsche, C. Erlenkämper, E. Moeendarbary, G. Charras, K. Kruse, Actin kinetics shapes cortical network structure and mechanics. Sci. Adv. 2e1501337 (2016).
69
M. Clarke, A. Müller-Taubenberger, K. I. Anderson, U. Engel, G. Gerisch, Mechanically induced actin-mediated rocketing of phagosomes. Mol. Biol. Cell 174866–4875 (2006).
70
F. S. Southwick, W. Li, F. Zhang, W. L. Zeile, D. L. Purich, Actin-based endosome and phagosome rocketing in macrophages: Activation by the secretagogue antagonists lanthanum and zinc. Cell Motil. Cytoskeleton 5441–55 (2003).
71
J. A. Spudich, S. Watt, The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J. Biol. Chem. 2464866–4871 (1971).
72
M. Winterhoff et al., The Diaphanous-related formin dDia1 is required for highly directional phototaxis and formation of properly sized fruiting bodies in Dictyostelium. Eur. J. Cell Biol. 93212–224 (2014).
73
G. Bertholdt, J. Stadler, S. Bozzaro, B. Fichtner, G. Gerisch, Carbohydrate and other epitopes of the contact site A glycoprotein of Dictyostelium discoideum as characterized by monoclonal antibodies. Cell Differ. 16187–202 (1985).
74
H. Troll et al., Purification, functional characterization, and cDNA sequencing of mitochondrial porin from Dictyostelium discoideum. J. Biol. Chem. 26721072–21079 (1992).
Information & Authors
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Published in
Proceedings of the National Academy of Sciences
Vol. 120 | No. 11
March 14, 2023
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Copyright
Data, Materials, and Software Availability
All study data are included in the article and/or SI Appendix. Constructs and mutants can be obtained from the corresponding author ([email protected]) upon reasonable request.
Submission history
Received: December 13, 2022
Accepted: February 3, 2023
Published online: March 10, 2023
Published in issue: March 14, 2023
Keywords
- actin
- formins
- macroendocytosis
- phagosome
- Rac
Acknowledgments
We thank Annette Breskott for technical assistance. This work was supported by a grant of the Deutsche Forschungsgemeinschaft to J.F. (FA330/13-1).
Author Contributions
S.K. and J.F. designed research; S.K. and J.F. performed research; A.J., C.L., and M.W. contributed new reagents/analytic tools; S.K. analyzed data; and S.K. and J.F. wrote the paper.
Competing Interests
The authors declare no competing interest.
Notes
This article is a PNAS Direct Submission.
Authors
Affiliations
Institute for Biophysical Chemistry, Hannover Medical School, 30625 Hannover, Germany
Institute for Biophysical Chemistry, Hannover Medical School, 30625 Hannover, Germany
Institute for Biophysical Chemistry, Hannover Medical School, 30625 Hannover, Germany
Moritz Winterhoff
Institute for Biophysical Chemistry, Hannover Medical School, 30625 Hannover, Germany
Institute for Biophysical Chemistry, Hannover Medical School, 30625 Hannover, Germany
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